The long-term objective of this project is to reduce coronary heart disease risk through a better understanding of the mechanisms of intestinal cholesterol absorption and the importance of cholesterol absorption in lipid removal of endogenous biliary cholesterol, but surprisingly few studies have actually measured cholesterol absorption in human subjects. Over the last several years this laboratory has developed cholesterol tracers and mass spectrometric methods that allow stable isotopic analysis of cholesterol absorption and metabolism in human subjects at high sensitivity. In this application we exploit these methods to address three important clinical questions. Specific Aim #1 tests the hypothesis that phytosterols (plant sterols) are much more active in reducing cholesterol absorption and LDL cholesterol levels than previously thought. This follows up our interim results which show that sitostanol, without effect in powdered form, has a nearly maximum effect at a dose of less than 300 mg when properly solubilized. Neomycin, an unrelated intra-intestinal cholesterol absorption inhibitor, will be compared to phytosterols and cooking oils with and without natural phytosterols will be examined. Specific Aim #2 tests the hypothesis that oxysterols, including those derived from phytosterols, are absorbed and biologically active. Interim work in rats demonstrated that dietary oxysterols are nearly quantitatively absorbed and also rapidly metabolized. Specific Aim #3 tests the hypothesis that cholesterol absorption is related to chronic dietary cholesterol, dietary fat, and plasma insulin level. Subjects will be fed from the GCRC kitchen and cholesterol absorption measured by isotopic methods. Successful completion of this project will provide new information in areas that have not received sufficient research attention. Knowledge of the importance of phytosterols, oxysterols and dietary determinants of cholesterol absorption will be used in supporting future dietary recommendations and research.